1. Cultured Peribulbar Dermal Sheath Cells Can Induce Hair Follicle Development and Contribute to the Dermal Sheath and Dermal Papilla 
Kevin J. McElwee, Sabine Kissling, Elke Wenzel, Andrea Huth, Rolf Hoffmann 
Journal of Investigative Dermatology 
Volume 121, Issue 6, Pages (December 2003)
DOI: /j x
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Isolation of the dermis derived hair follicle components. Whole mouse vibrissa follicles are dissected from donor tissue (a) and cut at a point where the tip of the DP is anticipated (arrows, b) to separate the peribulbar DSC and DP from the nonbulbar DS, root sheaths, and hair matrix (c). Hair matrix and sheath material is removed (d) and the DSC containing the DP (e) is everted (f). Remaining adherent keratinocytes and melanocytes are dislodged, the DP is cut (g) from the DSC (h), and each component is placed in separate cultures. In additional studies, the DSC was also cut from the collagen matrix before culture. The nonbulbar DS, closely adherent to the out root sheath, is separated (j) from the collagen capsule (i) for culture.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Alkaline phosphatase expression in the dermis-derived component of hair follicles in vivo and in vitro. Section through a C3H/HeJ mouse vibrissa exposed to alkaline phosphatase substrate (a). Expression (red) of alkaline phosphatase is apparent within the DP with greatest intensity in cells towards the apex of the DP above the line of Auber. A reduced alkaline phosphatase expression is apparent in cells of the DSC immediately below the DP as well as in DSC cells associated with the hair follicle bulb to a level approximately equal to the apex of the DP. The nonbulbar DS can be seen contiguous with the DSC but alkaline phosphatase is not expressed. Mouse guard hair (b) and pelage follicles (c) showed a similar presentation with strongest alkaline phosphatase expression toward the DP apex. Cells of the DSC immediately below the DP were moderately positive, whereas other DS cells were negative. Primary mouse cell cultures exposed to alkaline phosphatase substrate at 21 d, when the first cell passage was performed, showed that DS cells were consistently negative for alkaline phosphatase and exhibited a growth pattern of aligned, spindle-shaped cells typical of fibroblast-like cells (d). In contrast, DSC cells were slightly alkaline-phosphatase-positive, the growth pattern was more irregular, and cell morphology was less spindle-shaped (e). DP cells were somewhat more alkaline-phosphatase-positive and showed similar growth patterns to DSC cells (f). Both primary cultures of DSC and DP cells exhibited apparent clustering of cells (e, f) not observed in DS cell cultures. DS- (g, j, m), DSC- (h, k, n), and DP- (i, l, o) derived cell cultures exposed to alkaline phosphatase substrate 14 d after passages 1 (g–i), 2 (j–l), and 3 (m–o), when cells were confluent, illustrate maintenance of alkaline phosphatase expression in DP cells (i, l, o), whereas expression is lacking in passaged DS- and DSC-derived cell cultures. Bars (a–c), 100 μm (d–f), 100 μm, and (g–o) 2 mm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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4. Figure 3
Histologic presentation of amputated grafted mouse vibrissae and implantation of DP, DSC, and DS cells to Scid mouse ears and footpads. Of wild-type TgN-GFPX mouse vibrissae grafted to the backs of Scid mice, a proportion reformed DP and generated hair 6 mo after surgery (a–c). Morphologically, DP reformation occurred within the cut collagen capsule with apparent rearrangement of the collagen capsule to encompass the new DP (a) or, in hair follicles with smaller DP, without apparent collagen capsule production or rearrangement such that the DP was “exposed” through the cut end of the capsule (b). In one instance, apparent migration of DP and keratinocyte cells occurred such that the hair follicle bulb lay some distance below the cut end of the collagen capsule (c). Scid mouse ears implanted with Scid mouse derived DP cells (d, e, h) or DSC cells (f, g) exhibited multiple anagen-stage hair follicles and the presence of associated large hair fibers. The presence of larger DP was observed in association with hair follicles of greater disorientation (e, g), whereas hair follicles with smaller DP, but hair fiber of greater length than anticipated from natural mouse ear hair follicles (d, f), were more likely to be appropriately oriented. In addition, ears implanted with DP cells, as well as DSC cells, exhibited clusters of cells in the dermis not associated with epithelial structures (h). Footpads implanted with DP or DSC cells presented with large follicles (j). Nevertheless, mouse ears or footpads implanted with DS cells exhibited occasional patchy diffuse cells within the dermis, but no cell clustering or epithelium derived structures were observed (i, k). Bars, (a–c) 100 μm and (d–k) 100 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Hair growth from Scid mouse ears or footpads implanted with mouse-derived DP or DSC cells but not DS cells. Implantation of Scid mouse ears with TgN-GFPX mouse derived DP cells (left ear, arrowhead) resulted in hair fiber development significantly longer than that normally observed in non manipulated mouse ears or in ears implanted with DS-derived cells (right ear) after 6 mo (a). With DP (b) and DSC cell implantation the most common presentation involved several clusters of hair fibers (b, arrowhead). Nevertheless, a more even distribution and more appropriately oriented production of long hair fiber was observed in two DSC-cell-implanted ears (c). DP or DSC cells implanted to footpads produced comparatively few follicles for the same number of cells implanted, but long hairs emanating from the footpads (arrowheads) were apparent 6 mo after DP or DSC cell implantation (d), whereas DS-implanted footpads exhibited no gross morphologic changes (e).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Fluorescent cells within hair follicles of Scid mouse ears implanted with TgN-GFPX mouse-derived DP or DSC cells. The bulbs of large, anagen-stage hair follicles in Scid mouse ears implanted with TgN-GFPX mouse-derived DP cells demonstrated DP-containing fluorescent cells 6 mo after implantation (a). Ears implanted with TgN-GFPX mouse-derived DSC cells also contained anagen-stage hair follicles with fluorescent DP (b). While some DP structures exclusively contained fluorescent cells, hair follicles in ears implanted with DP (c) or DSC (d) cells also sometimes contained a combination of fluorescent and nonfluorescent cells. The number of fluorescent cells observed in an entire DP varied considerably with one hair follicle in DP-cell-implanted ears containing a single fluorescent cell (c), whereas other follicles, in this case from a DSC-cell-implanted ear, showed a mixture of several fluorescent and nonfluorescent cells (d). Fluorescent cells peripheral to the DP region, in DSC and DS areas of apparently induced hair follicles, could also be observed (a, b, e). Occasional anomalies were observed in DP- and DSC-implanted ears. In one DSC-cell-implanted ear, a single fluorescent cell cluster was associated with two separate hair follicles epithelium structures (f) with the bulb regions lying at a different orientation to the rest of the hair follicles. Fluorescence in DSC and DS regions, proximal to the follicle bulb, is due to a combination of fluorescent cells and nonspecific autofluorescence from deposited collagen. DP (g) and DSC (h) cells implanted to footpads also promoted hair follicle formation with associated fluorescent DP. Fluorescent cell clusters in the absence of associated epithelial structures could also be occasionally observed after DP or DSC implantation (i). Hair follicles in ears implanted with cells from wild-type TgN-GFPX did not demonstrate fluorescence within the DP or DSC (j). Mouse ear collagen (b, e, f) and keratinized hair shafts (e) are autofluorescent and nonspecific in these images. Bars, (a–j) 20 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions